Oral Microbiota from Periodontitis Promote Oral Squamous Cell Carcinoma Development via γδ T Cell Activation

ABSTRACT Oral squamous cell carcinoma (OSCC) is a fatal disease, and periodontitis is associated with OSCC development. However, the pathogenesis in the context of OSCC with periodontitis has not been fully understood. Here, we demonstrated that periodontitis promoted OSCC development, accompanied by alterations in the oral bacterial community and the tumor immune microenvironment. The oral microbiota from periodontitis maintained the dominant position throughout the whole process of OSCC with periodontitis, of which Porphyromonas was the most abundant genus. The oral microbiota from periodontitis could activate interleukin-17-positive (IL-17+) γδ T cells directly. The activated γδ T cells were necessary for the IL-17/signal transducer and activator of transcription 3 (STAT3) pathway and promoted M2-tumor-associated macrophage (TAM) infiltration in OSCC proliferation. Our data provide insight into the carcinogenesis of OSCC with periodontitis by outlining the tumor-associated immune response shaped by the oral microbiota from periodontitis. Thus, oral commensal bacteria and IL-17+ γδ T cells might be potential targets for monitoring and treating OSCC. IMPORTANCE The work reveals the role of the oral microbiota from periodontitis in carcinogenesis. Furthermore, our study provides insight into the pathogenesis of OSCC with periodontitis by outlining the tumor-associated immune response shaped by the oral microbiota from periodontitis, which might identify new research and intervention targets for OSCC with periodontitis.


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We thank the Editor for giving us this valuable opportunity to resubmit our current revised manuscript. We also addressed the Reviewers' comments by preparing point-by-point responses below.
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<b>Preparing Revision Guidelines</b> To submit your modified manuscript, log onto the eJP submission site at https://msystems.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary.

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• Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.

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We appreciate the Editor's reminder. We have prepared a separate letter entitled "Response to Reviewers" according to the requirements.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.

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We appreciate the Editor's reminder. We have prepared a comparison copy of the manuscript entitled "Marked-Up Manuscript" according to the requirements. All the modified text is highlighted in the revised manuscript.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.

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We appreciate the editor's reminder. We have verified all links to sequence records and made sure that each number retrieves the full record of the data.

Reviewer #1 (Comments for the Author):
This is a translation study investigating the role of periodontitis and associated microbiota in the promotion of OSCC development. The study shows oral microbiota from periodontitis patients promote RorgT cell mediated IL17-STAT3 pathway and tumor associated M2 macrophages as a fundamental pathway in OSCC development. While this study is significant, there are few weaknesses that need to be addressed.

Response:
We would like to thank the Reviewer for considering that our study was significant.
We have addressed the Reviewer's concerns by revising our manuscript and have responded to the comments point-by-point below.
1. The author state that ligation in mice did not affect tumor size development and proliferation without the inoculation of microbes from periodontitis patients. If so, is human oral bacteria more critical than native mice oral microbiota in OSCC development.

Response:
We appreciate the efforts of the reviewer in bringing this important issue to our attention. In our study, we performed a variety of interventions on mice, such as ligation alone, ligation with inoculation of healthy human oral bacteria (AON group), ligation with inoculation of periodontitis patient oral bacteria (OP/EOP/AOP group), and long-term antibiotic treatment (EOA/AOA group). Our experiment indicated that the changes in the mouse native oral microbiota and mouse immunity caused by ligation alone did not significantly contribute to the development of OSCC ( Supplementary Fig. 1c). In addition, we found that the healthy human oral bacteria also did not significantly affect the development of OSCC, but periodontitis oral bacteria did (Fig. 5). At the same time, 16S rRNA sequencing showed that the periodontitis oral bacteria dramatically altered the mice oral commensal bacterial community in OSCC, and some particular oral microbes from periodontitis could be the dominant "king" in the entire process of tumor development (Fig. 2). After comparison, we thought that although the human oral bacteria In conclusion, we believe that the human periodontitis oral bacteria need more attention in the study of oral squamous cell carcinoma. As suggested, we added the discussion, and please refer to the revised manuscript (Pages 20-21, Lines 281-289).
2. It's unclear if saliva from aggressive or chronic periodontitis patients were inoculated in mice?

Response:
We thank the reviewer for pointing this out, and we apologize for the missing information. The periodontitis saliva was from designated chronic periodontitis patients. The volunteers with periodontitis were all adults, and their teeth had obvious plaque, which was consistent with the degree of inflammation and destruction of periodontal tissue. The gingiva tissues were inflamed and bleeding on probing. The depth of the periodontal pocket was 4-6 mm, and X-ray films showed that the alveolar bone resorption exceeded 1/3 of the root length. As suggested, we have added detailed information in the Materials and Methods section. Please refer to the revised manuscript (Page 27, Lines 376-380).
3. It appears that advanced OSCC with Antibiotic (AOA) had the highest tumor burden and the authors attribute this to continuous use of antibiotic that might have resulted in the formation of drug-resistant bacterial community. The animals were treated with 3 days of antibiotics before oral bacterial inoculation. Three days of treatment will not lead to drug resistance.

Response:
We thank the reviewer for pointing this out, and we apologize for the missing information. The AOA and EOA groups were treated with 4Abx throughout the experimental period. The antibiotic drinking water treatment lasted for 29 days in the AOA and EOA groups ( Fig. 1b). 16S rRNA sequencing showed that the α diversity of the oral microbiome decreased under the 4Abx treatment (EOA and AOA groups) (Fig. 2a). As suggested, we have added 4. The study shows that periodontitis related microbiota plays a major role in OSCC development.
However, it is unclear which are the major pathogens involved. It's important to look into specific effects of P. gingivalis at least. Need to perform in vitro studies using P. gingivalis.

Response: We appreciate the reviewer's insightful commentary and valuable suggestions. Our
16S rRNA sequencing showed that Porphyromonas was the most abundant genus in the EOP (92.46%) and AOP groups (47.58%) (Fig. 2c). Fusobacterium, Neisseria, and Leptotrichia were more abundant in the AOP group ( Supplementary Fig. 2b). As suggested, we performed in vitro experiments to look into the specific effects of P. gingivalis. Although the proportion of γδ T cells in PBMCs did not increase after co-culturing with P. gingivalis, IL-17+ γδ T cells proliferated more than those in the group without P. gingivalis ( Supplementary Fig. 4a). At the same time, a significant increase in IL-17A was observed ( Supplementary Fig. 4b). When PBMCs and P. gingivalis were co-cultured with cancer cells, the increased expression of pSTAT3 in these cancer cells was observed ( Supplementary Fig. 4c) (7): 2652-2661). We are constantly monitoring the microbe. Our 16S rRNA sequencing showed that the α and β diversity of oral microbiota did not change significantly after inhibition of γδ T, but we will further analyze the oral microbiota by Metagenome and Metabolome Sequencing in the future. However, studies of the interplay between the resident microbiota and γδ T cells are limited, and additional mechanistic insights remain to be exploited in the future. In the Discussion section, we have added the latest research reports on the crosstalk between γδ T cells and microbiota. Please refer to the revised manuscript (Page 22, Lines 308-313) for details. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication. They will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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